Kohzy Hiramatsu

Central administration of galanin stimulates feeding behavior in chicks.

Galanin is recognized as one of the orexigenic peptides in the brain of mammals and fishes. The amino acid sequence of chicken galanin and its distribution in the brain are similar to those of mammals, suggesting that the brain galanin might be related to feeding regulation in chicks. The purpose of the present study was to investigate whether intracerebroventricular (ICV) injection of galanin affected feeding behavior of chicks (Gallus gallus). The injection of galanin increased food intake of layer and broiler chicks. We also found that the galanin-induced feeding behavior was attenuated in layer chicks by the co-injection of yohimbine and beta-funaltrexamine, which are the antagonists of adrenergic alpha-2 receptor and opioid mu-receptor, respectively. It is therefore possible that the orexigenic effect of galanin is mediated by these receptors.

Central administration of somatostatin stimulates feeding behavior in chicks.

The purpose of the present study was to determine if central administration of somatostatin influences feeding behavior in layer chicks. Five- to 7-day-old chicks that received intracerebroventricular (ICV) injections of 0.5 or 2 nmol somatostatin increased their food intake at 30 and 60 min after the injection, suggesting that central somatostatin serves as an orexigenic neuropeptide in chicks. This hypothesis was further supported since chicks ICV injected with 0.5 or 2 nmol cortistatin, which binds to somatostatin receptors, also had increased food intake at the same time. Somatostatin-associated feeding behavior was attenuated by co-administration of 20 nmol beta-funaltrexamine (an opioidergic mu-receptor antagonist) (to 31% of the orexigenic effect of somatostatin at 60 min after the injection) but not ICI-174,864 or nor-binaltorphimine (antagonists of opioidergic delta- and kappa-receptors, respectively). Co-administration of 13 nmol yohimbine, an adrenergic alpha-2 receptor antagonist, also attenuated the orexigenic effect of somatostatin (to 31% of the orexigenic effect of somatostatin at 60 min after the injection). These results suggest that somatostatin-associated feeding behavior is mediated by opioidergic mu- and adrenergic alpha-2-receptors in chicks.

Immunohistochemical localization of three different immunoglobulin classes in the Harderian gland of young chickens

The chicken Harderian gland (HG) was investigated using single immunohistochemical staining for one of the three different immunoglobulins (Igs) followed by Alcian blue/periodic acid Schiff (AB/PAS) staining and triple immunohistochemical staining for all of the Igs with hot water treatment. In the HG of 5-week-old chickens, IgG-containing plasma cells were more frequent than IgA- and IgM-containing cells. These numerous IgG-containing cells were predominantly accumulated in the central region of the stroma, whereas a small number of IgA- and IgM-containing cells were scattered in the peripheral region of the stroma. Also, the plasma cells containing PAS-positive Russell bodies (RBs) exhibited distinct immunoreactivity for one of the Igs, being inversely proportional to the intensity of PAS reaction. The RB-containing cells positive for IgA were more frequent than those positive for IgM, whereas those positive for IgG were very rare. The most distinct feature of the IgG-containing plasma cells was a PAS-positive globule located close to the nucleus. Triple immunostaining with hot water treatment simultaneously identified these three Igs in normal plasma cells and RB-containing ones in the stroma of the chicken HG.

Ultrastructural study on colocalization of glucagon-like peptide (GLP)-1 with GLP-2 in chicken intestinal L-cells.

ABSTRACT Colocalization of glucagon-like peptide (GLP)-1 with GLP-2 in L-cells was investigated in the chicken ileum by using double immunofluorescent and immunocytochemical techniques. Ultrastructural features of L-cells were also clarified in this study. L-cells showing immunoreactivity for both GLP-1 and GLP-2 were distributed in the whole ileum. They showed comma-like or flask-like shape and were located in epithelium of crypts and lower part of intestinal villi. L-cells showing GLP-1-immunoreactivity only were found in epithelium of lower and middle parts of intestinal villi. Transmission electron microscopy indicated that L-cells identified by colloidal gold-labeled immunocytochemistry were covered apically with microvilli, open-type and contained many secretory granules in their perikarya. These secretory granules without halo were round to oval in shape and showed moderate electron density. The longest and shortest diameters of secretory granules were 355 ± 62 nm (mean ± SD) and 287 ± 48 nm, respectively. Double labeling immunocytochemistry using two different sizes of particles (6 and 12 nm in diameter) of colloidal gold revealed that GLP-1 colocalized with GLP-2 in the same secretory granules. This study advances new morphological data about the endocrine system of the chicken small intestine.

Colocalization of NADPH-diaphorase with neuropeptides in the intrapancreatic neurons of the chicken

Colocalization of nitric oxide with neuropeptides was investigated in the chicken pancreas by use of double staining combined with the indirect immunofluorescence technique and histochemistry for NADPH-diaphorase, a specific marker for neural nitric oxide synthase. NADPH-diaphorase positive ganglia were easily detected in the interlobular connective tissue. Many NADPH-diaphorase positive ganglion cells also showed immunoreactivity for VIP (80.9%) or galanin (76.2%). Some ganglion cells showed enzyme activity only (about 20%). Very few neurons were NADPH-diaphorase negative, but immunopositive for VIP (2.0%) or galanin (3.7%). The present study provides evidence that nitric oxide colocates with VIP and galanin in the chicken pancreas.

Concerted and adaptive alignment of decorin dermatan sulfate filaments in the graded organization of collagen fibrils in the equine superficial digital flexor tendon

The equine superficial digital flexor tendon (SDFT) has a graded distribution of collagen fibril diameters, with predominantly small‐diameter fibrils in the region of the myotendinous junction (MTJ), a gradual increase in large‐diameter fibrils toward the osteotendinous junction (OTJ), and a mixture of small‐ and large‐diameter fibrils in the middle metacarpal (MM) region. In this study, we investigated the ultrastructure of the SDFT, to correlate the spatial relationship of the collagen fibrils with the graded distribution. The surface‐to‐surface distances of pairs of fibrils were found to be almost constant over the entire tendon. However, the center‐to‐center distances varied according to fibril diameter. Decorin is the predominant proteoglycan in normal mature tendons, and has one dermatan sulfate (DS) or chondroitin sulfate (CS) filament as a side chain which is associated with the surfaces of the collagen fibrils via its core protein. We identified a coordinated arrangement of decorin DS filaments in the equine SDFT. The sizes of the decorin DS filaments detected by Cupromeronic blue staining showed a unique regional variation; they were shortest in the MM region and longer in the MTJ and OTJ regions, and a considerable number of filaments were arranged obliquely to adjacent collagen fibrils in the MTJ region. This regional variation of the filaments may be an adaptation to lubricate the interfibrillar space in response to local mechanical requirements. The results of this study suggest that the MTJ region, which receives the muscular contractile force first, acts as a buffer for mechanical forces in the equine SDFT.

The influence of restricted feeding on glucagon-like peptide-1 (GLP-1)-containing cells in the chicken small intestine.

The influence of restricted feeding on the distribution of glucagon‐like peptide‐1 (GLP‐1)‐containing endocrine cells in the chicken small intestine was investigated using immunohistochemical and morphometrical techniques. This study demonstrated that the restricted feeding had an influence on the activity of GLP‐1‐immunoreactive cells in the chicken small intestine. There were differences in the localization and the frequency of occurrence of GLP‐1‐immunoreactive cells in the small intestine between control and restricted groups, especially 25% feed supply group provided with 25% of the intake during the adapting period. GLP‐1‐immunoreactive cells in the control chickens were mainly located in epithelium from crypts to the lower part of intestinal villi. Those in restricted groups, however, tended to be located from crypts to the middle part of intestinal villi. The frequency of occurrence of GLP‐1‐immunoreactive cells was lowest in the control group, medium in 50% feed supply group and highest in 25% feed supply group at each intestinal region examined in this study, that is, increased with the advancement of restricting the amount of feed supply. These data show that the quantity of food intake is one of signals that have an influence on the secretion of GLP‐1 from L cells in the chicken small intestine.

Histological analysis of glucagon-like peptide-1 receptor expression in chicken pancreas

Glucagon-like peptide-1 (GLP-1) released from intestinal L cells in response to nutrient ingestion inhibits both gastrointestinal emptying and gastric acid secretion and promotes satiety. The main biological effect of GLP-1 is the stimulation of insulin secretion (thereby fulfilling the criterion for an incretin hormone) in order to reduce blood glucose levels in mammalian species. Chicken GLP-1 receptor (cGLP-1R) has also been identified in various tissues by gene expression analysis. Although certain effects of GLP-1 in mammals and birds are consistent, e.g., inhibition of food intake, whether GLP-1 has the same insulinotropic activity in chickens as in mammals is debated. Moreover, the expression of cGLP-1R in chicken pancreatic B cells has not been reported. The localization of cGLP-1R and its mRNA in pancreatic islets is studied by triple-immunofluorescence microscopy and in situ hybridization. Triple-immunofluorescence microscopy with antisera against cGLP-1R, somatostatin and insulin or glucagon revealed that cGLP-1R protein was exclusively localized in D cells producing somatostatin in chicken pancreatic islets. The D cells were localized in peripheral areas of the pancreatic islets and cGLP-1R mRNA was detected in the same areas, indicating that cGLP-1R mRNA was also expressed in D cells. This is the first report to demonstrate that cGLP-1R is expressed by D cells, not B cells as in mammals. Our study suggests that chicken GLP-1 performs its insulinotropic activity by a different mode of action from that of the mammalian hormone.

Distribution of Glucagon-Like Peptide (GLP)-2-Immunoreactive Cells in the Chicken Small Intestine: Antigen Retrieval Immunohistochemistry

ABSTRACT An antigen retrieval method for immunohistochemical staining of glucagon-like peptide (GLP)-2-immunoreactive cells was investigated in the chicken small intestine. GLP-2-immunoreactive cells were observed as open-typed endocrine cells in the villous epithelium and crypts on both antigen retrieval agent-treated and untreated preparations. No obvious differences were detected in morphological features of GLP-2-immunoreactive cells between treated and untreated preparations. The frequencies of occurrence of GLP-2-immunoreactive cells, however, were significantly different in treated and untreated preparations: in the proximal and distal regions of jejunum and ileum obtained from untreated preparations, the frequencies of occurrence were 0.5 ± 0.2, 0.7 ± 0.1, 0.9 ± 0.2 and 1.5 ± 0.3, respectively (cell numbers per mucosal area: cells/mm2, mean ± SD), whereas those from treated sections were 14.7 ± 2.3, 19.8 ± 2.3, 23.5 ± 4.7 and 34.6 ± 4.9 cells/mm2, respectively. These data indicate that this antigen retrieval method is able to make immunoreactive GLP-2 available for detection and that GLP-2 may act as one of the common hormones secreted by L cells in the chicken small intestine.

Influences of protein ingestion on glucagon-like peptide (GLP)-1-immunoreactive endocrine cells in the chicken ileum

Influences of a specific dietary nutrient on glucagon-like peptide (GLP)-1-containing cells in the chicken intestine are not yet clear. Significance of dietary protein level on GLP-1-containing cells in the chicken ileum was investigated. Chickens fed control or experimental diets of varying protein levels were examined using immunohistochemical and morphometrical techniques. We show that the protein ingestion had an impact on the activities of GLP-1-immunoreactive cells in the chicken ileum. Weight gains declined with decreasing dietary crude protein (CP) levels, but no significant differences were detected in the daily feed intake and villous height. GLP-1-immunoreactive cells with a round or oval shape were frequently observed in the lower CP level groups (4.5% and 0%). Frequencies of occurrence of GLP-1-immunoreactive cells were 41.1 ± 4.1, 38.5 ± 4, 34.8 ± 3.1 and 34.3 ± 3.7 (cells/mm(2) , mean ± SD) for dietary CP level of 18%, 9%, 4.5% and 0% groups, respectively and significant differences were recognized between the control and lower CP level groups (P<0.05). Multiple regression analysis indicated a significant correlation between the daily protein intake and frequencies of occurrence of GLP-1-immunoreactive cells. The protein ingestion is one of the signals that influence GLP-1-containing cells in the chicken small intestine.